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Publisher Summary This chapter describes a method for making a vaccinia virus (VV) expression vector using the neomycin resistance gene in the recombinant plasmid pZVNEO and the selection of the recombinant VV with the antibiotic G418 sulfate (Geneticin, GIBCO-BRL). The foreign gene is being inserted behind the VV 7.5K promoter that has been used in most heterologous VV expression vectors because it contains early and late promoter elements and directs the efficient expression of the gene of interest throughout the viral life cycle. As an example, the chapter presents the generation of a recombinant VV expressing the vesicular stomatitis virus glycoprotein (VSV-G) protein that has been extensively used as a marker protein to study protein transport through the secretory pathway (Dascher and Balch, and references therein). The chapter also describes procedures for growing, isolating, and manipulating VV to familiarize the reader with basic techniques that are used in the process of isolating the recombinant virus. |
